Vinyl chloride (VC) is a toxic and carcinogenic priority pollutant that threatens drinking water quality in most industrialized countries. Kielhorn J., et al. (2000) Environ. Health Perspect. 108:579-588. A major source of environmental VC is due to transformation reactions acting on chlorinated solvents such as tetrachloroethene (PCE) and trichloroethene (TCE), which are abundant groundwater pollutants. Mohn W., et al. (1992) Microbiol. Rev. 56:482-507. Additional environmental VC pollution originates from landfills, PVC production facilities and abiotic formation in soils. Due to the extent of the problem, innovative and affordable technologies are needed to restore VC contaminated sites and guarantee drinking water safety.
Bioremediation approaches that rely on the activity of bacterial populations that use chlorinated compounds as growth-supporting electron acceptors (i.e., chlororespiration) have been used previously in the field (see, e.g., Ellis D., et al. (2000) Environ. Sci. Technol. 34:2254-2260; Major, D., et al. (2002) Environ. Sci. Technol. 36:5106-5116; Lendvay J., et al. (2003)Environ. Sci. Technol. 37:1422-1431). Bacterial populations useful in bioremediation include bacteria capable of reductive dechlorination and detoxification of VC to ethene. Such bacterial populations include members of the family Dehalococcoides, a deeply branching group on the bacterial tree most closely affiliated with the Chloroflexi. Cupples A., et al. (2003) Appl. Environ. Micobiol. 69:953-959. To facilitate the identification of bacterial populations responsible for dechlorination and detoxification of VC, 16S rRNA gene-based PCR approaches have been designed to detect and quantify members of Dehalococcoides. Such approaches have been helpful for assessing VC-contaminated sites, monitoring bioremediation efforts, and establishing cause-effect relationships between the presence of chlorinated compounds and the growth of specific strains of dechlorinating bacteria. Lendvay J., et al. (2003) Environ. Sci. Technol. 37:1422-1431.
Although 16S rRNA gene-based PCR approaches have been developed to detect and quantify members of Dehalococcoides, such approaches are limited in their applicability as Dehalococcoides strains with different dechlorination activities share similar or identical 16S rRNA gene sequences. He, J. et al. (2003) Nature 424:62-65. Examples of Dehalococcoides strains which demonstrate substantial similarities among 16S rRNA gene sequences, but distinct dechlorination activities include Dehalococcoides sp. strain CBDB1, which dechlorinates trichlorobenzenes, pentachlorobenzene and some polychlorinated dibenzodioxin congeners but failed to dechlorinate PCE and TCE (Adrian, et al. (2000) Nature 408:580-583), Dehalococcoides ethenogenes 195 and Dehalococcoides sp. and Dehalococcoides sp. strain FL2, which grow with polychlorinated ethenes as electron acceptors but cannot grow with VC, and Dehalococcoides sp. strain BAV1 which respires all DCE isomers and VC (He, J. et al. (2003) Nature 424:62-65). Despite their metabolic differences, these strains share 16S rRNA gene sequences with more than 99.9% similarity (based on the analysis of 1,296 aligned positions). He, J. et al. (2003) Appl. Environ. Microbiol. 65:485-495.
As a result of the high degree of identity among the 16S rRNA gene sequences of various Dehalococcoides populations, the identification of bacteria having different dechlorinating activities is difficult. There is, therefore, a need in the art for an improved means of identifying and characterizing reductively dechlorinating populations of bacteria. One such approach is to identify genes associated with the dechlorination of particular halogenated compounds, particularly genes encoding for reductive dehalogenases (RDases) capable of reductive dehalogenation of VC.
Gene sequences encoding for reductive dehalogenases involved in the partial reductive dechlorination of PCE and chlorinated aromatic compounds have been identified (see e.g., Magnuson, J., et al. (2000) Appl. Environ. Microbiol. 66:51441-5147). Functional genes involved in complete reduction of VC, however, have not been found. Alignment of known reductive dehalogenase amino acid sequences revealed low sequence identity (27 to 32%); although conserved stretches have been identified, e.g., a twin diarginine (RR) motif near the amino-terminus and two iron-sulfur cluster binding motifs near the C-terminus. Additionally, each of the identified RDase genes is associated with a B gene that encodes a hydrophobic protein with transmembrane helices believed to anchor the RDase to the membrane. Magnuson, J., et al. (2000) Appl. Environ Microbiol. 66:51441-5147. In Dehalococcoides, Sulfurospirillum (formerly Dehalospirillum), Dehalobacter and Desulfitobacterium, the B gene is located downstream of the PCE/TCE RDase genes. See e.g., Magnuson, J., et al. (2000) Appl. Environ. Microbiol. 66:51441-5147; Maillard, J., et al. (2003) Appl. Environ. Microbiol. 69:4628-4638; Suyama, A., et al. (2002) J. Bateriaol. 184:3419-3425. In cprA operons (ortho chlorophenol RDases) of Desulfitobacterium species an opposite arrangement was observed. Van de Pas, B., et al. (2003) J. Biol. Chem. 52:299-312.
Although gene sequences encoding reductive dehalogenases involved in the partial reductive dechlorination of PCE and chlorinated aromatic compounds have been identified, genes encoding enzymes capable of reductive dechlorination of vinyl chloride to ethene, have not been identified. Hence, there is a need in the art to identify functional genes associated with VC reductive dechlorination and in particular to identify and isolate reductive dehalogenase genes from dechlorinating bacteria and in particular those of the family Dehalococcoides. Additionally, there is a need in the art for a method of that identifies reductively dechlorinating populations of bacteria which overcomes the limitations of the identification methods of the prior art, and facilitate the monitoring of bioremediation by dechlorinating bacteria.